We have continued investigating mechanism of muscle contraction by correlating structural, mechanical and biochemical techniques applied to muscle fibers. In FY 1990, the following results are obtained: (A) Precursors to force generation: It has been proposed that during muscle contraction, crossbridges cycle between weak- and strong-binding states, and that force generation occurs as the result of a transition from the weak to the strong binding states. We have now shown that if the attachment of crossbridges to actin is blocked in the weak binding states, active force level is inhibited. The results show that within the cyclic interactions of crossbridge with actin, the crossbridges are required first to be bound to actin in the weakly bound states before generating active force, i.e. force-generating states are accessible only from attached weak-binding precursors. (B) Effect of calcium on the affinity of crossbridges to actin: According to the steric blocking model of calcium regulation in muscle, the affinity of crossbridges to actin is greatly affected by calcium. However, we have shown that in the presence of ATPgS, an ATP analog, calcium does not affect the affinity of crossbridge to actin. Rather, the kinetics of binding is affected. This finding is inconsistent with the steric blocking model. (C) Stiffness of the crossbridges in the radial direction: One mechanical property of crossbridges, i.e. stiffness, in the direction perpendicular to the fiber axis is shown to depend on the state of crossbridges. The differences in radial stiffness probably reflects structure differences in the binding of crossbridges to actin. This finding also raises the question whether axial stiffness is the same for all crossbridge states, which is commonly assumed.